ATCC BREAST CANCER RESEARCH RESOURCES
Transcript of ATCC BREAST CANCER RESEARCH RESOURCES
ATCC BREAST CANCER RESEARCH
RESOURCES
Fang Tian, Ph.D.
Lead Scientist, ATCC
April 24, 2014
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Who we are
• ATCC serves and supports the scientific
community with industry-standard products
and innovative solutions
• World’s leading biological resource center
and provider of biological standards
• Broad range of biological materials – Microorganisms
– Cell lines
– Derivatives
– Bioproducts
• Founded in 1925, ATCC is a non-profit
organization with headquarters in
Manassas, VA
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Outline
What do we know about breast cancer?
What’s new in breast cancer research?
What’s new about ATCC breast cancer cell lines?
ATCC breast cancer cells for animal models
ATCC primary breast cells and immortalized cells
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• Breast cancer is a malignant
form of cancer that develops in
breast tissue.
• Estimated new cases and deaths
in the United States in 2014:
Female Male
New Cases 232,670 2,360
Deaths 40,000 430
Breast cancer
Science 343:1454, March 28, 2014
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• Pathology subtype
• Stage classification
• Risk factor
• Clinical diagnostics
• Anti-breast cancer therapeutics
Special Issue | 28 March 2014
Breast Cancer
Breast cancer
Science 343:1454, March 28, 2014
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Molecular-based classification
• The categorization of breast tumors
based on hormone receptor and HER2
status
Top 21 most commonly mutated genes in
breast cancer
Gene All (%) Luminal (%) TNBC (%)
TP53 35 26 54
PIK3CA 34 44 8
GATA3 9 13 0
MAP3K1 8 11 0
MLL3 6 8 3
CDH1 6 8 2
USH2A 5 4 8 PTEN 3 3 3
RUNX1 3 4 0
MAP2K4 3 4 1
NCOR1 3 3 1
RB1 3 2 5
TBX3 2 3 1
PIK3R1 2 3 2
CTCF 2 2 1 NF1 2 2 1
SF3B1 2 2 0
AKT1 2 2 0
CBFB 1 2 1
FOXA1 1 1 1
CDKN1B 1 1 0
Polyak K, Filho OM. Cancer Cell 22(4):562, 2012.
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Outline
What do we know about breast cancer?
What’s new in breast cancer research?
What’s new about ATCC breast cancer cell lines?
ATCC breast cancer cells for animal models
ATCC primary breast cells and immortalized cells
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Large scale multicenter collaborative studies
Large scale initiatives
• The Cancer Genome Atlas (TCGA)
• International Cancer Genome Consortium (ICGC)
• Cancer Genome Project, Wellcome Trust Sanger Institute
• Collaborative Oncological Gene-environment Study (COGS)
Major outcomes
• Genetic landscape of breast cancer
• Genomic and clinical features of breast cancer
• New classification of breast cancer
• Genomic evolution of breast cancer
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Analysis of mutations across breast cancer
subtypes
The most significantly mutated genes in breast cancer as determined
by whole-exome sequencing
International Cancer Genome Consortium (ICGC). Nature 486: 405, June 21, 2012
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Point mutations
Copy number changes
Landscape of driver mutations in breast cancer
Each of the 40 cancer genes where a driver mutation or copy number
change has been identified is listed down the left-hand side
TP53
PIK3CA
ERBB2
MYC
FGFR1
Cancer Genome Project, Wellcome Trust Sanger Institute. Nature 486: 400, June 21, 2012
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Comprehensive molecular portraits of breast
cancer
Significantly mutated genes and correlations with genomic and clinical
features
The Cancer Genome Atlas Network. Nature 490: 61, October 4, 2012
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Genomic evolution in triple-negative breast
cancer
Population patterns of genomic aberrations in TNBC
Shah SP., et al. Nature 486: 395-399, June 21, 2012
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Genomic studies identify breast cancer risk loci
Collaborative Oncological Gene-Environment Study (COGS). Nature Genetics 392: 45(4), April 2013
Collaborative Oncological Gene-Environment Study (COGS). Nature Genetics 353: 45(4), April 2013
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Proposed new subgroup classification
Curtis C, et al.. Nature 486: 346-352, June 21, 2012
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Elucidation of novel breast cancer subgroups
Identification of 10 integrative subgroups with distinct copy number
profiles
Curtis C, et al.. Nature 486: 346-352, June 21, 2012
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Elucidation of novel breast cancer subgroups
The 10 integrative subgroups have distinct clinical outcomes
Curtis C, et al.. Nature 486: 346-352, June 21, 2012
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The life history of 21 breast cancer
A model for breast cancer
development over molecular time
Reconstruction of the
phylogenetic tree
Nik-Zainal S, et al. Cell 149, 994–1007, May 25, 2012
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Outline
What do we know about breast cancer?
What’s new in breast cancer research?
What’s new about ATCC breast cancer cell lines?
ATCC breast cancer cells for animal models
ATCC primary breast cells and immortalized cells
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ATCC breast cancer resource
• Complete list of ATCC breast cancer cell lines
– Human, mouse, rat, monkey, dog
• Breast cancer line by gene
• Variety of tumor cell panels
– By tissue
– By genetic alteration
– By in vivo model
• Paired tumor/normal cell lines
• Primary mammary epithelial cells
• hTERT immortalized mammary epithelial cells
• Mammary epithelial cell culture media
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ATCC breast cancer cell lines
Integratively designed Cell Panels
• Various tools
• Authenticated & high quality
• Convenience
• Value added
• Scientifically relevant
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ATCC Breast Cancer Cell Panels
Supportive materials If you are interested in
• Breast Cancer Cell Panel (ATCC® 30-4500K™)
• 45 breast cancer cell lines
Using a large number of cell lines to identify other rare or novel mutations/targets
• Triple-Negative Breast Cancer Cell Panels (ATCC® TCP-1001™, TCP-1002™, TCP-1003™)
Basic or translational research focused on triple-negative breast cancer
• Breast Cancer Biomarkers Cell Line Panel 1 (ATCC®
TCP-1004TM) Patient therapeutic treatment history or biomarker expression
• Breast Cancer Mouse Model Cell Panel (ATCC®
TCP-1005TM)
Breast cancer metastasis, in vivo mouse models of breast cancer, or the EGFR-MEK signaling pathways
• Breast Cancer p53 Hotspot Mutation Cell Panel (ATCC® TCP-2010TM)
p53 hotspot mutations, or characterization and validation data
• Genetic Alteration Cell Panels (ATCC® TCP-1027™ to TCP-1036™)
Significantly mutated genes, copy number changes, characterizations, and validation data
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Comprehensive Breast Cancer Cell Panel
(ATCC® 30-4500K™)
• 45 breast cancer cell lines
• Authenticated, high quality
• Cell line lot numbers traceable back to the same seed stock
• Includes commonly cited breast cancer cell lines
• Can be used in anti-breast cancer drug screening
• Can be used to identify rare or novel mutations/targets
• Provided with a CD containing signed certificates of analysis and
product sheets
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• Analyzed the gene expression profiles
from 21 breast cancer data sets and
identified 587 TNBC cases
• Identified 6 TNBC subtypes with unique
gene expression profiles and ontologies
• Assigned TNBC cell lines to subtypes
Triple-negative Breast Cancer Cell Panels
Background
Lehmann BD, et al. J Clin Invest 121(7): 2750-2767, 2011.
ATCC Triple-Negative Breast Cancer Cell Panels
Panel 1: Basal-like morphology
Panel 2: Mesenchymal & luminal
Panel 3: All 18 triple-negative breast cancer cell lines
Basal-like 1
Basal-like 2
Immunomodulatory
Mesenchymal like
Mesenchymal
stem like
Luminal AR
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Example of triple-negative breast cancer cell
lines
• Three triple-negative breast cancer cell panels
• Total of 18 cell lines
• Represent 6 identified subtypes
• Annotated with mutation data
ATCC® No. Name Subtype Tissue Tumor Source Histology Mutant
Gene Zygosity Gene Sequence
Protein
Sequence
HTB-132™ MDA-MB-
468 BL1 Breast
Metastasis;
pleural Adenocarcinoma
PTEN
RB1
SMAD4
TP53
Homozygous
Homozygous
Homozygous
Homozygous
c. 253+1G>T
c.265_2787del2523
c.1_1659del1659
c.818G>A
p.?
p.?
p.0?
p.R273H
CRL-2315™ HCC70 BL2 Breast Primary Ductal carcinoma PTEN
TP53
Homozygous
Homozygous
c.270delT
c.743G>A
p.F90fs*9
p.R248Q
CRL-2322™ HCC1187 IM Breast Primary Ductal carcinoma TP53 Homozygous c.322_324delGGT p.G108del
HTB-122™ BT-549 M Breast Ductal carcinoma Ductal carcinoma
PTEN
RB1
TP53
Homozygous
Homozygous
Homozygous
c.823delG
c.265_607del343
c.747G>C
p.V275fs*1
p.?
p.R249S
HTB-26™ MDA-MB-
231 MSL Breast Adenocarcinoma Adenocarcinoma
BRAF
CDKN2A
KRAS
NF2
TP53
Heterozygous
Homozygous
Heterozygous
Homozygous
Homozygous
c.1391G>T
c.1_471del471
c.38G>A
c.691G>T
c.839G>A
p.G464V
p.0?
p.G13D
p.E231*
p.R280K
HTB-131™ MDA-MB-
453 LAR Breast Carcinoma Carcinoma
CDH1
PIK3CA
Homozygous
Heterozygous
c.1913G>A
c.3140A>G
p.W638*
p.H1047R
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Breast Cancer Biomarkers Cell Line Panel
(ATCC® TCP-1004™)
Seven breast cancer cell lines isolated from variety of primary
and metastatic sites. Each cell line was annotated with pre-
operative therapeutics treatment and published biomarkers.
ATCC® No. Name Tumor
Source Pathology Age
Positive
markers
Negative
markers Other Significant Features Patient Treatment
CRL-1897™ UACC-812 Primary Infiltrating ductal
carcinoma 43 HER-2/neu
ER, PR,
EGFR, P-
glycoprotein
-
Vinblastine, Adriamycin,
Cytoxan, Cyclophosphamide,
Methotrexate, 5-fluorouracil
CRL-1902™ UACC-893 Primary Infiltrating ductal
carcinoma 57 HER-2/neu
ER, PR,
EGFR, P-
glycoprotein,
MASPIN
MASPIN promoter methylation has
been reported for this line None
CRL-2983™ UACC-3199
Metastasis;
axillary
nodes
Infiltrating ductal
carcinoma 58 EGFR
ER, PR, HER-
2/Neu Methylated BRCA-1 promoter
Cytoxan, Adriamycin, 5-
fluorouracil, Tamoxifen,
Mitoxantrone, Vinblastine
CRL-2988™ UACC-3133
Metastasis;
pleural
effusion
Ductal carcinoma 63 HER-2/neu,
BMP-3
ER (very low),
PR, EGFR,
MASPIN,
DSC3, BMP-2
MASPIN promoter methylation has
been reported for this line Surgery only
CRL-3127™ UACC-1179
Metastasis;
pleural
effusion
Adenocarcinoma 62 HER-2/neu
ER, PR,
EGFR,
MASPIN,
DSC3
P53 R213X mutation and MASPIN
promoter methylation have been
reported for this line
Adriamycin, Cytoxan,
Methotrexate, Tamoxifen
CRL-3166™ UACC-732
Metastasis;
pleural
effusion
Adenocarcinoma 35 HER-2/neu,
PR ER, EGFR
Drug resistant cell line to cyclin D
kinase 4/6 inhibitor and HER-2
inhibitors
Vinblastine, Adriamycin,
Cytoxan
CRL-3180™ UACC-2087
Metastasis;
pleural
effusion
Adenocarcinoma 53 EGFR
ER, PR, HER-
2/Neu,
vimentin,
MASPIN,
DSC3
P53 V216M mutation has been
reported in this cell line. It has also
been reported that the MASPIN
promoter is not methylated.
Cyclophosphamide,
Methotrexate, 5-flurouracil,
Thymidine phophorylase,
Tamoxifen
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p53 hotspot mutation cell panel (TCP-2010™) TP53 hotspot mutations
PI3K genetic alteration cell panel (TCP-1028™) PIK3CA hotspot mutations
PTEN genetic alteration cell panel (TCP-1030™) PTEN mutations and deletions
EGFR genetic alteration cell panel (TCP-1027™) EGFR mutations and amplification
EGFR genetic alteration cell panel (TCP-1027™) ERBB2 amplification
MYC genetic alteration cell panel (TCP-1035™) MYC mutations and amplification
FGFR genetic alteration cell panel (TCP-1034™) FGFR1, FGFR2 amplification
ATCC molecular
signature cell panels Features
New tools relevant to recent studies
TP53
PIK3CA
PTEN
EGFR
ERBB2
MYC
FGFR1
Top genes reported
in breast cancer
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p53, guardian of the genome
Jie Xu et al., Nature Chemical Biology 7: 285-295, 2011
p53 gain of function mutation
Breast cancer p53 hotspot mutation cell panel
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Molecular mechanisms of breast cancer
Polyak K, Filho OM. Cancer Cell 22(4):562, 2012.
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Molecular Signature Cell Panels
TKR
EGFR&ERBB2
FGFR1&2
HGFR/MET PI3K
AKT
PTEN
RAS
BRAF
ERK MYC
p53
Growth factor receptors
Transcription factors PI3K-AKT pathway
MAPKs pathway
Site mutation
Copy number change
Gene expression
Protein expression
Signaling pathway
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Breast cancer cell lines in EGFR cell panel
C-D: ERBB2, G-H, merged with F-actin/ Hoechst
ATCC®
number Cell line name gene
EGFR copy
number
variation
ERBB2 copy
number
variation
Tumor
source Histology
HTB-132™ MDA-MB-468 EGFR Amplification − Breast Adenocarcinoma
HTB-19™ BT-20 EGFR Amplification − Breast Carcinoma
HTB-20™ BT-474 ERBB2
− Amplification Breast Ductal carcinoma
HTB-27™ MDA-MB-361
ERBB2 − Amplification Breast Adenocarcinoma
HTB-25™ MDA-MB -175 VII WT control cell line Breast Ductal carcinoma
CRL-10317™ MCF10A WT control cell line Normal breast Normal
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Breast cancer cell lines in EGFR cell panel
ATCC®
number Cell line name Gene
EGFR copy
number variation ERBB2 copy
number variation Tumor source
Histology
Other mutations in related signaling
pathway
HTB-
132™ MDA-MB-468 EGFR Amplification − Breast Adenocarcinoma
HTB-19™ BT-20 EGFR Amplification − Breast Carcinoma MAPK1 H61Q
HTB-20™ BT-474 ERBB2 − Amplification Breast Ductal carcinoma PIK3CA H1047R
HTB-27™ MDA-MB-361
ERBB2 − Amplification Breast Adenocarcinoma PIK3CA E545K; PIK3CA K567R;
• Captured the key molecular features in breast cancer
• Verified mutation status, gene expression, and protein expression
• Represented the genetic complexity observed in clinical patients
HTB-25™ MDA-MB -175 VII WT control cell line Breast Ductal carcinoma
CRL-10317™ MCF10A WT control cell line Normal breast Normal
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Paired tumor/ normal cell lines
Tumor-derived cell lines matched to normal cell
lines obtained from the same patient
Tumor cell lines
tumor source Pathology Name ATCC® No
Normal pairing
tissue source Pathology Name ATCC® No.
Metastasis: lymph node Ductal carcinoma HCC10008 CRL-2320™ B lymphoblast Normal HCC1007 BL CRL-2319™
Mammary gland Ductal carcinoma Hs574.T CRL-7345™ Skin Normal Hs574.Sk CRL-7346™
Mammary gland Ductal carcinoma Hs578T HTB-126™ Mammary gland Normal Hs578Bst HTB-125™
Mammary gland Ductal carcinoma HCC1954 CRL-2338™ B lymphoblast Normal HCC1954 BL CRL-2339™
Mammary gland Ductal carcinoma HCC38 CRL-2314™ B lymphoblast Normal HCC38 BL CRL-2346™
Mammary gland Ductal carcinoma HCC1143 CRL-2321™ B lymphoblast Normal HC1143 BL CRL-2362™
Mammary gland Ductal carcinoma HCC1187 CRL-2322™ B lymphoblast Normal HCC1187 BL CRL-2323™
Mammary gland Ductal carcinoma HCC1395 CRL-2324™ B lymphoblast Normal HCC1395 BL CRL-2325™
Mammary gland Ductal carcinoma HCC1599 CRL-2331™ B lymphoblast Normal HCC1599 BL CRL-2332™
Mammary gland Ductal carcinoma HCC1937 CRL-2336™ B lymphoblast Normal HCC1937 BL CRL-2337™
Mammary gland Ductal carcinoma HCC2218 CRL-2343™ B lymphoblast Normal HCC2218 BL CRL-2363™
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Outline
What do we know about breast cancer?
What’s new in breast cancer research?
What’s new about ATCC breast cancer cell lines?
ATCC breast cancer cells for animal models
ATCC primary breast cells and immortalized cells
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Breast cancer cell line in vivo models
Subcutaneous models • HTB-30™ SK-BR-3
• HTB-132™ MDA-MB-468
Orthotopic models • HTB-20™ BT474
• HTB-26™ MDA-MB-231
• HTB-22™ MCF-7
• HTB-130™ MDA-MB-436
• HTB-131™ MDA-MB-453
Commonly used human breast cancer line xenografts
Commonly used murine breast cancer model • CRL-2539™ 4T1
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Revisit mouse breast cancer cell lines
• New hope of antibody therapy – Anti CTLA-4
– Anti PD-1
• Combination therapy – Immunotherapy + molecular targeted
therapy
• Need for appropriate in vivo models – Immunocompetent mouse model
Cancer immunotherapy-
Scientific breakthroughs from 2013
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Nat Rev Mol Cell Biol.
Nov 2008
Dr. Philip Leder
ATCC® No. Designation
CRL-3063™ Eph4Ev
CRL-3069™ B-MEKDD 116
CRL-3071™ Eph4 1424
CRL-3209™ Eph4 1424.1
CRL-3210™ Eph4 1424.2
CRL-3086™ M158
CRL-3090™ NF639
CRL-3092™ Ac 711
• Mouse mammary cell lines
• MEK mutation
• EGFR pathway
• Oncogenes in cell
transformation
Metastatic
tumor in kidney
Metastatic
tumor in lung
Primary
mammary tumor
Express constitutively activated MEK1.
Glu-Glu epitope-tagged MEK1
phosphorylation site mutant (MEKDD)
Express empty vector
CRL-3063
Eph4Ev
Eph4, immortalized
mouse mammary
gland epithelial cell line
CRL-3069™
B-MEKDD 116
CRL-3071™
Eph4 1424
CRL-3209™
Eph4 1424.1
CRL-3210™
Eph4 1424.2
Breast Cancer Mouse Model Cell Panel
(ATCC® TCP-1005™)
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Transgenic mouse models and cell lines have
been used in signal pathway studies and drug
discovery
Cancer Cell 2: 29-42, 2001; Oncogene 16: 737 – 746, 1998
mammary tumor
mammary tumor
mammary tumor
MMTV-c-myc transgenic mouse
MMTV-c-neu transgenic mouse
Zeta-Glovin-v-Ha-ras transgenic mouse
Ha-Ras
EGFR2
Myc myc
neu
Ha-ras
CRL-3086™
M158
CRL-3090™
NF639
CRL-3092™
Ac 711
Breast Cancer Mouse Model Cell Panel
(ATCC® TCP-1005™)
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Outline
What do we know about breast cancer?
What’s new in breast cancer research?
What’s new about ATCC breast cancer cell lines?
ATCC breast cancer cells for animal models
ATCC primary breast cells and immortalized cells
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Primary Cells – weighing the pros and cons
• Prepared directly from tissue
• Physiologically-relevant
• Low risk for phenotypic or genotypic drift
• Sourcing of tissue may be difficult and
expensive to establish
• Isolation conditions may be difficult and
expensive to establish
• Small yield
• Limited culture life
CONs
PROs
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Epithelial Tissue
• Corneal Epithelial Cells
• Prostate Epithelial Cells
• Small Airway Epithelial Cells
• Bronchial/Tracheal Epithelial Cells
• Renal Proximal Tubule Epithelial Cells
• Renal Cortical Epithelial Cells
• Renal Mixed Epithelial Cells
• Keratinocytes – Neonatal Foreskin Epidermal Keratinocytes
– Adult Epidermal Keratinocytes
• Melanocytes – Neonatal Foreskin Epidermal Melanocytes
– Adult Epidermal Melanocytes
Normal human primary cells from ATCC
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Primary Mammary Epithelial Cells
(ATCC® PCS-600-010)
CK14 CK18
myoepithelial luminal
Cell Specifications:
• Cryopreserved at low passage (P2)
• Tested for: – High post-thaw viability
– Growth to ≥ 15 PDL
– Free from microbial contamination.
Primary Mammary epithelial cells display CK14-positive
(myoepithelial marker) and CK18-positive (luminal marker).
ATCC® PCS-600-010 Normal, Human
Primary Mammary Epithelial Cells
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Telomerase hTERT
HPV-16 E6
Myc T58A
p53/p21 SV40T
HPV-16 E6
p16/pRB HPV-16 E7
CDK4
Bmi-1
Other Methods Feeder culture (3T3)
Rho-associated kinase inhibitor (Y-27632)
Physiological Oxygen (2-5%)
Roads to cell immortalization
Plasmids and Reagents ATCC® No.
hTERT MBA-141™
SV40-T VRMC-3™
HPV-16 E6/E7 CRL-2203™, 45113D™
CDK4 MGC-19704™, MGC-4678™, MGC-3719™
Bmi-1 81582D™, MGC-12685™
3T3 Feeder Cells CCL-92™, 48-X™
ROCK Inhibitor Y-27632 ACS-3030™
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hTERT-HME1 [ME16C] (ATCC® CRL-4010™)
ATCC® CRL-4010™
stained with pancytokeratin mAb
(green) and Hoechst dye (blue)
• The human mammary epithelium hTERT-HME1
[ME16C] (ATCC® CRL-4010™) cell line was derived
from normal primary mammary epithelial cells.
• Human telomerase reverse transcriptase (hTERT)
immortalized cell lines combine the in vivo nature of
primary cells with the traditional cell line's ability to
survive continuously in vitro.
• hTERT-HME1 cells have served as normal controls in
several studies that sought to unravel the molecular
mechanism of breast cancer pathogenesis.
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Summary
• Breast cancer is the leading cause of cancer-related mortality in women.
• New disease classifications, relevant signaling pathways, and genetic regulators of breast cancer have been identified over the past decade.
• To continue facilitating progress in basic research and drug discovery, ATCC provides comprehensive breast cancer research resources including a large number of breast cancer cell lines, various tumor cell panels with in-depth genetic alteration and molecular profiles, useful cell lines for developing in vivo animal model, as well as primary cells and immortalized mammary epithelial cells. These tools can be used to address the newest identified genomic and clinical features of breast cancer subtypes.
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